Process of fermentation of chlortetracycline and tetracycline antibiotics



United States Patent 7 Claims. 61. 195-114 This application is a continuation-in-part of copending application Ser. No. 141,461, filed on July 24, 1961 and now abandoned.

The present invention relates to an improved process for the production, by submerged fermentation, of N,N- dibenzylethylenediamine (DBED) and N,N-lower-dialkoxydibenzylethylenediamine alkaline earth metal complexes of chlortetracycline and tetracycline, as well as to an improved process of fermentation of chlortetracycline and tetracycline, these being easily freed in pure state from those complexes.

More particularly, the invention relates to the addition of an effective amount of a compound selected from the group consisting of N,N-dibenzylethylenediamine and a N,N'-1ower-dialkoxydibenzylethylene diamine to the broth to be fermented, enhancing the formation of chlortetracycline and tetracycline, thus representing a more economical process than hitherto described.

These new derivatives possess favorable pharmacological properties such as more lasting blood levels, and, due to their very low solubility in aqueous medium, they are useful for the isolation of the active substances from any fermented broth containing tetracycline or chlortetracycline.

The use of N,N'-dibenzylethylenediamine together with magnesium ions [for the recovery of tetracyclines has been claimed in Czech Patent No. 91,397, without disclosing specifically the individual complexes or their physical constants, biological activity or pharmaceutical usefulness.

The U.S. Patent 2,712,517 discloses the use of precursors, i.e., compounds metabolically convertible into the tetracycline molecules, exerting an enhancing action on the yield in the fermentation of tetracycline type antibiotics.

In accordance with the present invention, the addition to the fermentation broth of small quantities of an organic compound structurally not related to the tetracycline molecule provokes surprisingly an increase in the yield of the antibiotic by up to 2000 mcg./ml., yielding pharmaceutically useful derivatives of chlortetracycline and tetracycline according to the kind of strain of the fermentation microorganism used, from which the antibiotic chlortetracycline and tetracycline can be liberated. It has been found that surprisingly the yield of the known microorganisms producing chlortetracycline as well as those producing tetracycline will be increased by addition of N,N-dibenzylethylenediamine to the culture media, independently of the species used.

The object of the present invention is to prepare the pharmaceutically useful DBED complexes of chlortetracycline and tetracycline in pure state by fermentation and to provide improved methods of fermentation of chlortetracycline and tetracycline which are suitable for commercial use.

In accordance with the present invention, there is provided a process for fermentation of a conventional broth, to which a small amount of a compound selected from the group consisting of N,N-dibenzylethy1enediamine and a N,N'-lower-dialkoxydibenzylethylenediamine is added portionwise before and during fermentation, carried out under submerged aerobic conditions at a temperature range of 26 to 30 C. by strains of microorganisms having the capacity to produce antibiotics selected from the group comprising chlortetracycline and tetracycline until a substantial amount of antibiotic activity is conferred upon the broth, comprising furthermore the consecutive steps of preparing the DBED alkaline earth metal complexes in a pure state suitable for pharmaceutical use by recrystallizing the crude DBED complexesobtained by precipitation at a pH range of 7-9.5 from the previously acidified and filtered broth4from dimethylformamide water solution and liberating the chlortetracycline and tetracycline antibiotics from the respective crude complexes by oxalic acid extraction at a pH range of 1 to 1.6, and the successive and conventional crystallization of the corresponding base of chlortetracycline and tetracycline at a pH range of 3.5 to 5.5.

The fermenting microorganism is a strain having the capacity to produce an antibiotic included in the group of chlortetracycline and tetracycline. Strains of Streptomyces aureofaciens (ATCC 10762, ATCC 13908, ATCC 14896, NCIB 8234, NCIB 9122), Streptomyces viridifaciens (ATCC 11989), Streptomyces psammoticus (IGUP S 4623/33) and Streptomyces sayamaensis are suitable for industrial purposes. However, the preferred process is to use Streptomyces lusitainus for the production of chlortetracycline and its DBED complex in a conventional chloride ion containing medium and in a chloride ion free medium for the production of tetracycline and its DBED complex. Strepzomyces lusitarrus has been described in Antibiotics and Chemitherapy-1962. (pp. 661-668, American Society for Microbiology, Ann Arbor, Miclr, 1963) and the original soil isolate is deposited in the American Type Culture CollectionWashington,under No. ATCC 15842; an industrially useful selected live strain descen-dent of the former in deposited in the National Collection of Industrial BacteriaAberdeen, under N0. NCIB 9451. It should be noted that, the industrial strains being highly sensitive and unstable, preserved cultures are to be submitted to the conventional selection procedures prior to industrial use.

Another advantage of the present invention-besides the increased yields of fermentation-lies in the fact that water is the sole solvent used during the whole process where chlortetracycline and tetracycline base is prepared. Thus the fire hazard is practically excluded and the most economical solvent is used, whereas in the preparation of the pharmaceutical grade quality of the DBED complexes an organic solvent is used only in the final step.

The culture media conventionally used for chlortetracycline and tetracycline fermentation are suitable. The examples given here below illustrate the composition of such media.

In order to increase yields, DBED is added to the broth before and during fermentation under sterile conditions as a water soluble salt selected from the group comprising diacetate and di-lactate in a total amount of 0.1 to 2.5 grs. per litre divided into 3 to 6 fractions. A suitable schedule for portionwise addition is at 0, 48, 72, and hours. A preferred way of performing the invention is to add increasing portions of DBED to the broth proportionally to the activity formed. Thus, whereas the first and second fractions are very small amounting to quantities of 20 to 100 mgs./litre, the quantities of successive additions are increased. However, the total amount added should not surpass at the last addition the molecular ratio of 1 mole of DBED and 2 moles of the antibiotic formed. After -l50 hours the rate of activity formation diminishes and fermentation is stopped.

The fermentation is carried out by known conventional processes under submerged aerobic conditions at a temperature range of 26-28 C.

Example 1 illustrates the increase in yield of antibiotic crystallizes out in pure pharmaceutical useful state. The formation using various strains of various species in paraddition of sodium hydrosulfite to the solution improves allel fermentations run without addition of DBED. The the color of the final product. The thus obtained chlorvalues indicate that the variation in the increase of yield tetracycline and tetracycline correspond to the specificadepends on the properties of a particular strain rather tion given in US. Pharmacopea XV and XVI respecthan on those of a given species. Previous laboratory tively. scale fermentations enable those skilled in the art to find Example 1 the optimum amount of the fractions of DBED to be add- 40 ml. ortions of a culture medium of the followin ed and the time intervals for a particular strain, in the p g light of the illustration given in the examples. composmon' The fermented broth, once fermentation is stoppedas com SteeP llquor 25 known in the art-is acidified with 12% sulphuric acid, PeanPt meal 25 filtered and the DBED complex is precipitated by adjust- Calclum calibonaie 8 ing the pH to 79.5. As the conventional culture media soluble Starch (hlgh fluldlty) 61 used in the present process for the tetracycline antibiotic 02 4 fermentation contain calcium salts, the excess of calcium NH4c1 ions is sequestered by adding ethylenediamine tetraace- 4 2 tate. Addition to the broth of an antioxidant, such as soc0012 (1005 dium hydrosulfite or formaldehyde sulfoxylate sodium be- Lard 011 "8 30 fore precipitation of the complex, improves the quality vF6504 0-001 and color of the antibiotic. p Water 100 The crude DBED calcium Complexes 9 chlortefracy' are placed in 300 cc. Erlenmeyer flasks and sterilized at cline and tetracycline thus formed are purlfied by dissolvfor 5 minutes, the PH being 62 fl i i ing them in the minimuf'n amount of dlmethylformamlde tion. The flasks are inoculated with 1 cc. of a 24-hour-old IleCeSSaTY- The Solution 15 filterd and the DBED Complex 25 culture of the respective microorganism, the composition precipitated by adding to it 5 times volume/volume of of this Seed medium being as follows; water under stirring. The precipitated complex is filtered, washed with water and dried at 45 C. under vacuum. Com Steep hquor 10 Sucrose grs 10 The centesimal composition of the anhydrous complex compound corresponds to the molecular composition of C8003 1 DBED-CTC -Ca and DBED-TC -Ca. However, com- (NH4)4HPO4 2 plexes of other composition have been isolated specially KH2PO4 2 when the amount of DBED was less than 1 mole for ev- MgSO4-7H2O ery two moles of antibiotic, having a composition after Tap Water 1000 isolation of 1:423. and incubated in a rotary shaker at 28 C. A sterile con- The in vitro activity of the complexes corresponds to centrated aqueous DBED acetate solution is added before that of their tetracycline antibiotic content. In vivo they and during fermentation as indicated in the annexed table exercise sustained therapeutically useful blood levels for excepting controls and the antibiotic content of broth is periods over 36 hours. The blood levels were determined determined after 140 hours by standard U.V. assays. The

in rabbits after a single intramuscular dose of an amount 40 values obtained demonstrate the effectiveness of addition of DBED-TC -Ca suspended in saline equivalent to 100 of DBED to the culture media. mg./kg. of tetracycline hydrochloride and as a control 100 mg./kg. of pure tetracycline hydrochloride in buff- Examp 1e 2 ered saline were injected in another group of rabbits. 300 litres sterilized culture medium of the composition as specified in Example 1 are inoculated with 1500 ml. of Megs. or tetracycline per ml. of plasma a 24 hours old seed culture of the composition as specified Eggs; after admlmstraflon in Example 1 of a strain of Streptomyces Iusitanus NCIB 1 3 s 24 30 9451. A sterile concentrated aqueous solution of DBED 6 [H L3 L6 L6 L1 di-acetate is added to the broth in portions of 20, 50, 70, TO.HC1 6 1.0 1.8 1.4 0.3 230, 630, 1500 mg./l1tre at O, 48, 72, 96, 120, 134 hours of submerged aerobic fermentation in an adequate fer- The complexes are also suitable as active ingredients for tati t n-k equipped i h a stirrer at 26 C ili dermatological ointments. air being injected at a pressure of 0.5 kg./cm. After 145 m e de DBED complexes obtained after a1kahours the activity of the DBED.CTC .Ca complex thus line precipitation chlortetracycline and tetracycline ref r d TABLE Antibiotic content after 140 hours in me .1111. ex ress d l d hl id Amount of DBED acetate added g/ p e as W we or e of to the broth expressed in mg./litre Chlortetracycline Tetracycline S. aureo- S. aurco- S. luxitan'us S. Zusitamts S. sayama S. sam- S. m'rtdi- S.

0 48 72 96 faciens faciena NCI B 9451, NCIB 9451, ensis mo zz'cus facz'em fa iti? N RRL 2209 N RRL 2209, selection No. selection No. selection N0. selection N o. AICC 11989, prevailing selected AX/502 AX/801 34 7 selection No. tetracycline Hours after inoculation gg g M 99 ggg ggz strain No. M 97 spectively can be isolated in pure state by suspending the is equivalent to 10.9 gr./litre of chlortetracycline hydrost1 ll wet complex in Water and extracting the antibiotic chloride. Then 1 gr./litre of sodium hydrosulfite and 1.5 with a 10% oxalic acid solution at pH 1.5. The acid solugr./litre of ethylenediamine tetraacetate sodium are tion is filtered and its pH raised to 6-7 and then decreased added. After separating the mycelium from the broth by to 4-55, when the corresponding tetracycline antibiotic 75 filtration of the broth acidified with diluted sulphuric acid at pH 1.0, the activity decreases to 7.6 gr./litre due to dilution. The pH is then adjusted to 8.2 by adding ammonia water. The precipitate thus obtained is filtered, washed with water and well drained. The still wet precipitate weighs 7.9 kg. consisting of crude DBED-CTC Ca complex. Subsequently, it is dissolved in a minimum amount of dimethylformamide, the impurities are filtered off and the complex is precipitated in pure state by adding 5 volumes of water to the dimethylformamide solution. The precipitate is filtered, washed with water and dried at 45 C. under vacuum. The desired pure chlortetracycline DBED calcium complex thus obtained is characterized by the following absorption maxima and minima in U.V. light:

Max. 230 m (EF Z 244); 265 III/L (Ei'fi 270); 305 m (E85... 121) 340 mp. (E1 3... 14.6); 375 m (Er 3,, 164) and min. 240 mu (ER'Z 214) 305 mu (Eifg 111) 344 m 1 2... 1

In a parallel control fermentation, the addition of DBED has been omitted. The activity of broth corresponded to 9.1 gr./ litre expressed in chlortetracycline hydrochloride.

Example 3 300 litres sterilized culture medium of the composition as specified in Example 1 are inoculated with 1500 ml. of a 24 hours old seed culture of the composition as specified in Example 1 of a selected strain descendent of Streptomyces aureofaciens NRRL 2209. A sterile concentrated aqueous solution of DBED diacetate is added to the broth in portions of 20, 50, 70, 230, 630 mg./litre at 0, 48, 72, 96, 120 hours of submerged aerobic fermentation in an adequate fermentation tank equipped with a stirrer at 28 C., sterilized air being injected at a pressure of 0.5 kg./ cm. After 135 hours the activity of the DBED 'CTC Ca complex thus formed is equivalent to 6 gr./litre of chlortetracycline hydrochloride. Then 1 g./'litre of sodium hydrosulfite and 1.5 g./litre of ethylenediamine tetraacetate sodium are added. After separating the mycelium from the broth by filtration of the broth acidified with diluted sulphuric acid at pH 1.0, the activity decreases to 3.8 gr./litre due to dilution. The pH is then adjusted to 8.2 by adding 10% ammonia water. The precipitate thus obtained is filtered, washed with water and well drained. The still wet precipitate weighs 4.3 kg. consisting of crude DBED'CTC -Ca complex. Subsequently, the precipitate is reslurried in 10 litres of water and acidified with a 10% aqueous oxalic acid solution to pH 1.6, and the formed calcium oxalate and the impurities are filtered off. The pH of the clear filtrate is adjusted to pH 6.5 with 10% sodium hydroxide and 0.6 gr./litre of sodium hydrosulfite is added. The pH is then decreased to 5.5 with concentrated hydrochloric acid and cooled to +5 C. under stirring. The chlortetracycline base crystallizes out in pure state. It is filtered, washed with Water and dried in vacuum. Purity 98.2%.

Example 4 One proceeds as in Example 2, but the microorganism used is a recently selected strain of Streptomyces viridifacie'ns (ATCC 11989). After 145 hours, the activity of the DBED tetracycline calcium complex, thus formed, is equivalent to 5.4 gr./litre of tetracycline hydrochloride. 1 gr./litre of sodium hydrosulfite and 1.2 gr./-litre of ethylenediamine tetraacetate are then added. After separating the mycelium from the broth by filtration of the broth acidified with diluted sulphuric acid at pH 1.5, the activity decreases to 3.7 gr./litre due to dilution. The pH is then adjusted to 9.5 by adding 10% ammonia water. The precipitate, thus obtained, is filtered, washed with water and well drained. The still wet precipitate weighs 4.1 kg. consisting of crude DBED-TC -Ca complex. Subsequently, it is dissolved in a minimum amount of dimethylfomnamide and the impurities are filtered off. The complex is then precipitated in pure state by adding 5 volumes of water to the solution. The precipitate is then filtered, washed with water and dried under vacuum at 45 C., yielding the desired pure DBED tetracycline calcium complex, which exhibits the following maxima and minima in its U.V. absorption curve:

Max. 358 mu (Ei g 250); 270 m (Ei'fi m (Elfi 252) and min. 300 m (Elfi (Ei'ff 131.5) in N/ H SO4 Antibiotic activity 880 meg/mg.

Example 5 6000 litres of a sterilized dechlorinated culture medium 308) and 215 148); 233 mp.

per litre of chloride ion free water are inoculated with litres of a 24 hours old seed culture of a recently selected strain of streptomyces lusitanus (NCIB 9451). The seed medium is of the composition as specified in Example 1, but the corn steep liquor and water used are free of chl0- ride ions. A sterile concentrated aqueous solution of DBED di-acetate is added to the broth in portions of 20, 50, 70, 230, 600, 1000 mg./litre at 0, 48, 72, 96, 120 and hours of submerged aerobic fermentation at 28 C., sterilized air being injected at a pressure of 0.5 kg./cm. After 148 hours, fermentation is stopped, the broth is acidified to pH 1.5 with diluted sulphuric acid and filtered. 1.2 gr./litre of ethylenediamine tetraacetate and 0.9 gr./ litre of sodium hydrosulfite are added to the filtrate and the pH is raised to 9.5 with 12% ammonia. After stirring for 3 hours, the precipitate formed is filtered and Washed with water. The wet precipitate is reslurried in 200 litres of water and acidified with a 10% aqueous oxalic acid solution to pH 1.6, and the formed calcium oxalate and the impurities are filtered off. The pH of the clear filtrate is adjusted to pH 6.5 with 10% sodium hydroxide and 0.6 gr./litre of sodium hydrosulfite is added. The pH is then decreased to 5.5 with concentrated hydrochloric acid and cooled to +5 C. under stirring. The tetracycline base crystallizes out in pure state. It is filtered, washed with water and dried in vacuum. Purity 97.9%. Overall yield 69%.

Example 6 One proceeds as in Example 5, but instead of DBED di-acetate one adds the same amounts of N,N-di-(p-methoxybenzyl)-ethylenediamine di-lactate to the broth. Purity of the tetracycline thus obtained 99.1%. Overall yield 66%.

What is claimed is:

1. A process for the production of an antibiotic substance selected from the group consisting of chlortetracycline, tetracycline, N,N-dibenzylethylenediamine and N,N lower dialkoxydibenzylethylenediamine alkaline earth metal complexes of chlortetracycline and tetracycline, which comprises the steps of growing, under submerged aerobic conditions, a culture of a species of Streptomyces which produces an antibiotic selected from the group consisting of chlortetracycline and tetracycline in an aqueous nutrient medium containing assimilable carbon, nitrogen and mineral salts, in the presence of a member selected from the group consisting of N,N'-dibenzylethylenediamine, N,N' dialkoxydibenzylethylenediamine and the Water soluble salts thereof in at least an effective amount, the total of which does not exceed half a mole per mole of the said antibiotic content formed to form the said complexes until a substantial antibiotic activity is imparted to the broth, and recovering said antibiotic substance from said culture medium.

2. A process for the production of N,N'-dibenzylethylenediamine chlortetracycline calcium complex, which comprises the steps of growing, under submerged aerobic conditions, a culture of a species of Streptomyces which produces chlortetracycline in an aqueous nutrient medium containing assimilable carbon, nitrogen and mineral salts, in the presence of N,N'-dibenzylethylenediamine di-acetate, added in fractions before and during fermentation the total amount of which does not exceed half a mole per mole of the chlortetracycline content formed, until a substantial antibiotic activity is imparted to said solution, precipitating the said complex from the filtered acidified broth at pH 6 to 8.5, and recrystallizing the precipitate from dimethylformamide-water.

3. A process for the production of chlortetracycline, which comprises the steps of growing, under submerged aerobic conditions, a culture of a species of Streptomyces which produces chlortetracycline in an aqueous nutrient medium containing assimilable carbon, nitrogen and mineral salts, in the presence of N,N-dibenzylethylenediamine di-acetate, added in fractions before and during fermentation the total amount of which does not exceed half a mole per mole of the chlortetracycline content formed, until a substantial antibiotic activity is imparted to said solution, precipitating the N,N'-dibenzylethylenediamine chlortetracycline calcium complex from the filtered acidified broth at pH 6 to 8.5, extracting the wet precipitate with an aqueous oxalic acid solution at pH 1.5 and crystallizing chlortetracycline at a pH from 3.2 to 5.5.

4. A process for the production of N,N'-dibenzylethylenediamine tetracycline calcium complex, which comprises the steps of growing, under submerged aerobic conditions, a culture of a species of Streptomyces which produces tetracycline in an aqueous nutrient medium containing assimilable carbon, nitrogen and mineral salts, in the presence of N,N-dibenzylethylenediamine di-acetate, added in fractions before and during fermentation the total amount of which does not exceed half a mole per mole of the tetracycline content formed, until a substantial antibiotic activity is imparted to said solution, precipitating the said complex from the filtered acidified broth at pH 6 to 9.5, and recrystallizing the precipitate from dimethylformamide-water.

5. A process for the production of N,N'-dibenzylethylenediamine tetracycline calcium complex, which cornprises the steps of growing, under submerged aerobic conditions, a culture of a species of Streptomyces which produces tetracycline in an aqueous chloride ion free nutrient medium containing assimilable carbon, nitrogen and mineral salts, in the presence of N,N-dibenzylethylenediamine (ii-acetate, added in fractions before and during fermentation the total amount of which does not exceed half a mole per mole of the tetracycline content formed, until a substantial antibiotic activity is imparted to said solution, precipitating the said complex from the filtered acidified broth at pH 6 to 9.5, and recrystallizing the precipitate from dimethylformamide-water.

6. A process for the production of tetracycline, which comprises the steps of growing, under submerged aerobic conditions, a culture of a species of Streptomyces which produces tetracycline in an aqueous nutrient medium containing assimilable carbon, nitrogen and mineral salts, in the presence of N,N'-dibenzylethylenediamine di-acetate, added in fractions before and during fermentation the total amount of which does not exceed half a mole per mole of the tetracycline content formed, until a substantial antibiotic activity is imparted to said solution, precipitating the N,N'-dibenzylethylenediamine tetracycline calcium complex from the filtered acidified broth at pH 6 to 8.5, extracting the wet precipitate with an aqueous oxalic acid solution at pH 1.5 and crystallizing tetracycline at a pH from 3.2 to 5.5.

7. A process for the production of tetracycline, which comprises the steps of growing, under submerged aerobic conditions, a culture of a species of Streptomyces which produces tetracycline in an aqueous chloride ion free nutrient medium containing assimilable carbon, nitrogen and mineral salts, in the presence of N,N'-dibenzylethylenediamine di-acetate, added in fractions before and during fermentation the total amount of which does not exceed half a mole per mole of the tetracycline content formed, until a substantial antibiotic activity is imparted to said solution, precipitating the N,N-dibenzylethylenediamine tetracycline calcium complex from the filtered acidified broth at pH 6 to 8.5, extracting the wet precipitate with an aqueous oxalic acid solution at pH 1.5 and crystallizing tetracycline at a pH from 3.2 to 5.5.

References Cited UNITED STATES PATENTS 2,712,517 7/1955 Gourevitch et a1 1 14 FOREIGN PATENTS 91,397 8/ 1959 Czechoslovakia.

MAURICE W. GREENSTEIN, Primary Examiner. 

1. A PROCESS FOR THE PRODUCTION OF AN ANTIBIOTIC SUBSTANCE SELECTED FROM THE GROUP CONSISTING OF CHLORTERTRACYCLINE, TETRACYCLINE, N,N''-DIBENZYLETHYLENEDIAMINE AND N,N'' - LOWER - DIALKOXYDIBENZYLETHYLENEDIAMINE ALKALINE EARTH METAL COMPLEXES OF CHLORTETRACYCLINE AND TETRACYCLINE, WHICH COMPRISES THE STEPS OF GROWING, UNDER SUBMERGED AEROBIC CONDITIONS, A CULTURE OF A SPECIES OF STREPTOMYCES WHICH PRODUCES AN ANTIBIOTIC SELECTED FROM THE GROUP CONSISTING OF CHLORETRACYCLINE AND TETRACYCLINE IN AN AQUEOUS NUTRIENT MEDIUM CONTAINING ASSIMBILE CARBON, NITROGEN AND MINERALS SALTS, IN THE PRESENE OF A MEMBER SELECTED FROM THE GROUP CONSISTING OF N,N-DIBENZYLETHYLENEDIAMINE, N, N''-DIALKOXYDIBENZYLETHYLENEDIAMINE AND THE WATER SOLUBLE SALTS THEREOF IN AT LEAST AN EFFECTIVE AMOUNT, THE TOTAL OF WHICH DOES NOT EXCEED HALF A MOLE PER MOLE OF THE SAID ANTIBIOTIC CONTENT FORMED TO FORM THE SAID COMPLEXES UNTIL A SUBSTANTIAL ANTIBIOTIC ACTIVITY IS IMPARTED TO THE BROTH, AND RECOVERING SAID ANTIBIOTIC SUBSTANCE FROM SAID CULTURE MEDIUM. 